Method for detecting rheumatoid arthritis employing the CB10 peptide of mammalian type II collagen as an antigen

ABSTRACT

An highly specific and sensitive approach to detecting antibodies to collagen in a biological sample entails contacting the sample with an antigen that comprises the CB10 peptide of mammalian type II collagen, or with an antibody-binding fragment of that peptide, under conditions conducive to formation of an antibody-antigen complex, which then is detected.

FIELD OF THE INVENTION

[0001] This invention relates to the diagnosis of rheumatoid arthritis,a chronic disease of the joints and other tissue that is a serioushealth problem worldwide. In particular, the present invention isdirected to providing a predictive test for rheumatoid arthritis in itsearly stages which shows high specificity and sensitivity, enablingprompt and accurate diagnosis and hence effective treatment withappropriate drugs. Such early treatment can limit irreversible jointdamage which is known to occur within the first few years or even monthsafter the onset of rheumatoid arthritis.

BACKGROUND OF THE INVENTION

[0002] Rheumatoid arthritis is the most serious of the rheumaticdisorders in terms of population prevalence, potential for crippling andmorbidity, and life-shortening effects. The disease is characterised bythe symmetrical inflammation of multiple joints (polyarthritis). It mostfrequently affects the small joints of the hands and feet, butinflammation can occur in virtually any joint including spinal joints.Pain, stiffness and swelling of the joints are the main symptomaticfeatures, resulting in loss of function. Damage to the joints leads toserious deformities and functional impairment. Apart from the effects onthe joints, rheumatoid arthritis may be associated with a wide range ofextra-articular features affecting various organs, such as the heart,blood vessels, lungs and kidneys. Although these extra-articularfeatures are most common in the case of serious forms of rheumatoidarthritis, they may also provide the first symptom of the disease. Thereis at present no reliable cure for rheumatoid arthritis. Treatment isessentially directed towards alleviating the discomfort caused by thesymptoms and arresting the progression of the disease. Sometimes,however, the disease appears to resolve spontaneously, or in response toone or other of the drug regimens currently employed.

[0003] Rheumatoid arthritis generally appears after puberty. Theprevalence rises with age, and it is 2-3 times more frequent among womenthan men. The prevalence of definite rheumatoid arthritis is between1-2% in the majority of white populations (Hochberg, M C, 1981), and thetreatment of patients with rheumatoid arthritis consumes a significantcomponent of the health care budget.

[0004] Rheumatoid arthritis is included among the autoimmune diseases.Many authors assume that exposure to an infectious agent, bacterium orvirus, can initiate rheumatoid arthritis in individuals with a geneticpredisposition to the disease. The actual disease is generated by anabnormal reaction of the immune system, which then plays a central rolein the progression of articular damage and extra-articular lesions.Since no infectious agent has in fact been convincingly implicated inthe disease, rheumatoid arthritis may be a spontaneously occurringautoimmune process, in which the primary response is to an autoantigeniccomponent of the joint itself, rather than to an extrinsic infectiousagent.

[0005] The idea that rheumatoid arthritis is an autoimmune response to acomponent of cartilage is traced back to Steffen and Timpl (1963) whofirst showed antibodies to collagen in rheumatoid arthritis and proposedthat rheumatoid arthritis results from an autoimmune response to thecollagen molecule present in cartilage now know to be type II collagen.This idea is strongly supported by the observation that immunizationwith type II collagen induces an arthritis with similarities to humanrheumatoid arthritis in appropriate strains of rats, mice or primates(Courtney et al 1980, Trentham et al, 1977).

[0006] In human rheumatoid arthritis, autoantibodies to native anddenatured type II collagen are detectable in the serum and synovialfluid of up to 30% of patients according to data derived fromcross-sectional studies on patients with generally long-standing disease(Morgan et al, 1987; Terato et al, 1990; Rowley et al, 1992). However,the importance of such antibodies to type II collagen has long remainedcontroversial in view of their low frequency in most reported studies,the lack of correlation between antibody levels and disease status(Clague et al, 1980b Stuart et al, 1983; Collier et al, 1984; Morgan etal, 1989; Stockman et al, 1989) and the reported presence of theseantibodies in a range of disease other than rheumatoid arthritis (Clagueet al, 1980; Trentham et al, 1981; Gioud et al., 1982; Rosenberg et al,1984; Charriere et al., 1988; Choi et al., 1988; Rowley et al., 1988,1992). As mentioned, most of the positive associations of about 30%between antibodies and disease have been based on patients withrheumatoid arthritis of long duration. More recently, several studieshave shown that the frequency of autoantibodies to type II collagen maybe as high as 60-75% in patients tested very early in the course ofrheumatoid arthritis, and levels of autoantibody tend to fall as thedisease progresses to levels ascertained in the earlier cross-sectionalstudies (Pereira et al, 1985; Fujii et al, 1992, Cook et al, 1994,1996). Accordingly, it has been proposed that antibodies to collagenwill provide a useful predictive marker of early rheumatoid arthritisand particularly so for those patients in whom rapid progression tojoint destruction will occur.

[0007] The main structural proteins of the connective tissue in the bodyare collagens, of which at least 19 genetically different types have sofar been described (Brodsky and Shah, 1995). The types of collagen foundin a specific tissue are related to the function of the tissue, and theyhave specific distributions within individual tissues. Articularcartilage in mature joints contains a number of different collagentypes, of which type II collagen is the most abundant. It is the majorfibrous collagen in all hyaline cartilage and represents 80-90% of thecollagen content. Its role is to build up a fibrous network which,together with proteoglycans and hyaluronan, creates an extremely strongstructure with the capacity to withstand high pressures (Heinegard andPaulsson, 1987). Type II collagen is a highly conserved molecule betweenspecies. It consists of 3 identical α chains, and is moderatelyglycosylated (Miller, 1985). It is restricted to cartilage and few othertissues, these being the vitreous humour of the eye, and intervertebraldiscs, in contrast to the more universal distribution of type I and IIIcollagens (Gay et al., 1980).

[0008] In general, the basic structure of all native collagen consistsof three polypeptide α-chains in the form of a triple helical domain(s)with repeating glycine-X-Y triplets, in which X is often proline and Yis often hydroxyproline (Piez, 1982). Hydroxyproline is essential forthe formation of hydrogen bonds that stabilize the helix. Each α-chainis coiled into a tight left-handed helix which averages about 3 aminoacid residues per twist. Three α-chains coil about one another in aright-handed manner to create a 300 nm long, 1.5 nm thick, superhelixwhich is stabilized by hydrogen bonds formed between the α-chains. About25 to 30 amino acid residues are required on each chain to complete oneturn of the superhelix. Heat denaturation of collagen molecules at 45°C. leads to unfolding of the triple helix to display the linear sequenceof amino acids along the length of the individual α-chains.

[0009] Collagen is stabilized by the formation of covalent cross-links.Two kinds of cross-links are formed in the collagen fibre, intra- andinter-molecular. Cross-link formation involves enzymatic conversion oflysine and hydroxylysine residues, to allysine and hydroxyallysinerespectively, by peptidyl lysine oxidase. Allysine and hydroxallysinereact with each other, or with lysine, spontaneously, to form aldol andaldimine condensation products. No enzyme catalysis is required for thisprocess, only the physical proximity of the appropriate side chain(Miller, 1985). Cross-links are formed between a modified lysyl orhydroxlysyl residue in the telopeptide region and a hydroxylysyl residuein the conserved triple helical region, and the amount of cross-linkingincreases with the age of the individual.

[0010] The intact triple helical domain of collagen is resistant toalmost all enzymes except mammalian collagenase which cleaves mostcollagens into a three-quarter length TcA fragment and a one-quarterlength TcB fragment. However, upon heating, the triple helix isdenatured and thus susceptible to non-specific proteinases. Cleavage ofcollagen with cyanogen bromide, which cleaves at the carboxyl terminalof methionine residues, produces a unique set of peptides, called CBpeptides, which can be purified by a combination of ion-exchange andmolecule sieve chromatography (Piez, 1976). For type I collagen, CBpeptides have been numbered using rat collagen as the standard, and fortype II collagen, CB peptides have been numbered using chick collagen asthe standard. When a methionine cleavage site is missing as may occur incollagen of another species, the numbers designating the 2 uncleavedpeptides are both used, separated by a comma (see FIG. 1). Separatedα-chains and CB peptides are able to renature, under appropriateconditions, to their original triple-helical conformation (Terato et al,1985). CB-peptides of type II and type III collagen are particularlyamenable to renaturation when separated, because in each type the threeα-chains are identical (Terato et al, 1985, Werkmeister and Ramshaw,1991).

[0011] As noted above, the type II collagen molecule consists of threechains wound in a triple helix, and techniques do not yet exist thatallow production of a “native” type II collagen molecule by geneticengineering from cDNA constructs. For this reason, measurement ofantibodies to collagen requires the use of collagen purified fromcartilage, whether of human or animal origin, or purified fromchondrosarcoma cell lines. However, since most healthy people have lowlevels of natural antibody to collagen in their serum, perhapsrepresenting a response to dietary collagens, tests designed to measureantibodies to collagen in rheumatoid arthritis using the entire moleculeof purified collagen will not necessarily give disease-specific results.Thus, it is preferable to identify regions of the collagen molecule thatreact specifically with antibodies in sera of patients with rheumatoidarthritis.

[0012] In the work leading to the present invention, the regions of thecollagen molecule (epitopes) that are recognized by antibodies tocollagen have been examined by Western blotting, using cyanogen bromidedigests of collagen, whereby the collagen molecule is cleaved atmethionine residues, to produce a number of CB peptides (see FIG. 1).Most studies on antibodies to CB peptides of collagen in rheumatoidarthritis disclose responses to multiple peptides (Terato et al, 1990;Buckee et al, 1990; Rowley et al, 1992; Cook et al, 1994). In the past,however, no significant difference has been found in the pattern ofrecognition of CB peptides by patients with rheumatoid arthritis orother diseases (Rowley et al, 1992), except that antibodies to type IIcollagen in the inflammatory disease of cartilage, relapsingpolychrondritis, react with one of the smaller CB polypeptides ofcollagen, CB9,7, which is rarely recognized by patients with rheumatoidarthritis (Terato et al, 1990).

SUMMARY OF THE INVENTION

[0013] In a first aspect, the present invention provides a method forthe detection of antibodies to collagen in a biological sample from apatient, the method comprising contacting said biological sample with anantigen comprising the CB10 peptide of mammalian type II collagen, or anantibody-binding fragment or variant thereof, for a time and underconditions for an antibody-antigen complex to form, and then detectingsaid complex.

[0014] It will, of course, be apparent to persons skilled in this artbased on the disclosure herein that the detection of an antibody-antigencomplex in accordance with the method of this invention is an indicatorof rheumatoid arthritis in the patient, and whilst there is no simpledefinitive test for rheumatoid arthritis, the method of this inventionenables the detection of antibodies to collagen as a predictive markerof early rheumatoid arthritis.

[0015] The antigen used in the method of the present invention comprisesthe CB10 peptide of mammalian type II collagen, or an antibody-bindingfragment or variant thereof, from any suitable source, particularlybovine or human type II collagen. The CB 10 peptide, or antibody-bindingfragment thereof, may be produced by cleavage of the whole collagenmolecule with Cyanogen bromide, followed by purification of the CB10fraction, as is well known in the art. Alternatively, however, a similarfraction of the collagen molecule may be prepared by other proteinchemistry techniques, including solid phase and other protein synthesistechniques.

[0016] The method of the present invention utilises an immunoassayformat, including for example an ELISA format, which is well known topersons skilled in this art.

[0017] The present invention also extends in another aspect to adiagnostic kit for the detection of antibodies to collagen in abiological sample from a patient, which comprises, in compartmentalform, an antigenic preparation comprising the CB10 peptide of mammaliantype II collagen, or an antibody-binding fragment or variant thereof,and means for detection of a complex formed between said antigen andantibodies to collagen in said biological sample.

[0018] Preferably, the biological sample is a serum, plasma or wholeblood sample from the patient. Preferably also, the biological sample istaken from a human patient.

[0019] Throughout this specification and the claims which follow, unlessthe context requires otherwise, the word “comprise”, and variations suchas “comprises” and “comprising”, will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The diagnosis of rheumatoid arthritis in its early stages isdifficult. Until characteristic signs of cartilage and bone destructionappear on X-rays, rheumatoid arthritis cannot be reliably distinguishedfrom certain limited forms of arthritis which are of differentpathogenesis. Traditionally aspirin and/or other non-steroidalanti-flammatory drugs are given as a first-line of treatment forrheumatoid arthritis, and more potent agents are added if and when thedisease deteriorates. However this sequence of treatment does notoptimally halt progressive bone and joint destruction. Because thegreatest loss of function occurs early in the disease, progressivelywithin the first four years, rheumatologists now believe that giving themost powerful drugs early in the course of treatment is more likely toprevent the destruction that leads to crippling disability. However,since these potent disease-modifying drugs can have significant adverseeffects, a marker to distinguish those patients who will developcrippling disease is urgently needed. As noted previously, there havenow been several studies that suggest that antibodies to collagen arepresent very early in rheumatoid arthritis, before the diagnosis isclearly established, and before irreversible joint destruction will haveoccurred. Accordingly, a method of measuring collagen antibodies thatcan be reliably performed in routine diagnostic laboratories would haveconsiderable utility.

[0021] Most current immunoassays to detect to the entire type IIcollagen molecule antibodies do not show sufficient specificity andsensitivity for the diagnosis of rheumatoid arthritis, noting that theseantibodies may be demonstrable in other diseases, or even in healthyindividuals, or may be lacking in cases of established rheumatoidarthritis particularly cases of long duration.

[0022] In accordance with the present invention, it has now been shownthat the use of the particular CB peptide, CB10 of mammalian type IIcollagen, or an antibody-binding fragment or variant thereof, instead ofthe entire collagen molecule, provides for an immunoassay to detectantibodies to collagen that greatly increases the specificity andsensitivity of the immunoassay as a diagnostic test for rheumatoidarthritis.

[0023] An “antibody-binding fragment” or variant thereof contemplated bythe present invention includes a fragment of the CB10 peptide of amammalian type II collagen comprising at least 50% of the sequence ofsaid CB10 peptide. Preferably, the percentage of the CB10 sequence is atleast about 60%, more preferably at least about 70%, even morepreferably at least about 80% and still even more preferably at leastabout 90% or greater. Such a fragment could be prepared from collagenpurified from cartilage, or expressed as a recombinant protein in anappropriate expression system, or derived by any other means. Mostpreferably said fragment exhibits the helical structure of the CB10peptide and retains the ability to bind to collagen antibodies. Suitableantibody-binding fragments may be identified by simple trials toascertain their ability to bind to collagen antibodies.

[0024] The present invention also extends to the use of variants of theCB10 peptide or antibody-binding fragment thereof as described above,particularly substitution variants which are conservative in nature andresult from replacing one amino acid in the naturally-occurring CB10amino acid sequence with another having similar structural and/orchemical properties, such as the replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, or a threonine witha serine. Other suitable variants of the naturally-occurring sequenceinclude insertion or deletion variants, typically of from about one tofive amino acids. Suitable variants will preferably exhibit the helicalstructure of the CB10 peptide and retain the ability to bind to collagenantibodies, and may be identified by simple antibody-binding trials.

[0025] The detection of the presence of antibodies to collagen inaccordance with the present invention may be accomplished in a number ofimmunoassay procedures, such as by ELISA procedures. A wide range ofimmunoassay techniques is available as can be seen by reference tostandard immunoassay textbooks. These, of course, include bothsingle-site and two-site or “sandwich” assays of the noncompetitivetypes, as well as the traditional competitive binding assays.

[0026] Sandwich assays are among the most useful and commonly usedassays and are favoured for use in the present invention. A number ofvariations of the sandwich assay technique exist, and all are intendedto be encompassed by the present invention. Briefly, in a typical assayto detect antibodies in a sample, an unlabelled antigen is immobilizedon a solid substrate and the sample to be tested brought into contactwith the bound antigen molecule. After a suitable period of incubation,for a period of time sufficient to allow formation of anantibody-antigen complex, a second antibody such as anti-human IgG,labelled with a reporter molecule capable of producing a detectablesignal, is then added and incubated, allowing time sufficient for theformation of another complex of antibody:antigen:labelled antibody. Anyunreacted material is washed away, and the presence of the antibody tobe detected in the sample is determined by observation of a signalproduced by the reporter molecule. The results may either bequalitative, by simple observation of the visible signal, or may bequantitated by comparing with a control sample containing known amountsof the antibody to be detected. Variations on this assay include asimultaneous assay, in which both sample and labelled antibody are addedsimultaneously to the bound antigen. These techniques are well known tothose skilled in the art, including any minor variations as will bereadily apparent.

[0027] In the typical sandwich assay, antigen is immobilised, forexample by being either covalently or passively bound to a solidsurface. The solid surface is typically glass or a polymer, the mostcommonly used polymers being cellulose, polyacrylamide, nylon,polystyrene, polyvinyl chloride or polypropylene. The solid supports maybe in the form of tubes, beads, discs, or microplates, or any othersurface suitable for conducting an immunoassay. The binding processesare well-known in the art and generally consist of cross-linking,covalent binding or physical adsorption. The immobilised antigen is thenwashed in preparation for the test sample. An aliquot of the sample tobe tested is then contacted with the immobilised antigen and incubatedfor a period of time sufficient (e.g. 2-40 minutes) and under suitableconditions (e.g. 25° C.) to allow binding of any antibody to collagenpresent in the sample. Following the incubation period, the immobilisedantigen with any bound antibody is washed and dried, and incubated witha second antibody specific for the bound antibody, for exampleanti-human IgG. The second antibody is linked to a reporter moleculewhich is used to indicate the binding of the second antibody to theantibody:immobilised antigen complex.

[0028] By “reporter molecule” as used in the present specification, ismeant a molecule which, by its chemical nature, provides an analyticallyidentifiable signal which allows the detection of antigen-boundantibody. Detection may be either qualitative or quantitative. The mostcommonly used reporter molecules in this type of assay are eitherenzymes, fluorophores or radionuclide-containing molecules (i.e.radioisotopes), or chemiluminescent molecules. In the case of an enzymeimmunoassay (EIA), an enzyme is conjugated to the second antibody,generally by means of glutaraldehyde or periodate. As will be readilyrecognized, however, a wide variety of different conjugation techniquesexist, which are readily available to the skilled artisan. Commonly usedreporter enzymes include horseradish peroxidase, glucose oxidase,beta-galactosidase and alkaline phosphatase, amongst others. Thesubstrates to be used with the specific enzymes are generally chosen forthe production, upon hydrolysis by the corresponding enzyme, of adetectable colour change. It is also possible to employ fluorogenicsubstrates, which yield a fluorescent product rather than thechromogenic substrates noted above. In all cases, the enzyme-labelledantibody is added to the first antibody-antigen complex, allowed tobind, and then the excess reagent is washed away. A solution containingthe appropriate substrate is then added to the complex ofantibody:antigen:labelled antibody. The substrate will react with theenzyme linked to the second antibody, giving a qualitative visualsignal, which may be further quantitated, usually using aspectrophotometric instrument, to give an indication of the amount ofantibody which was present in the sample. “Reporter molecule” alsoextends to use of cell agglutination or inhibition of agglutination,such as red blood cells on latex beads, and the like.

[0029] Alternately, fluorescent compounds, such as fluorescein andrhodamine, may be chemically coupled to antibodies without alteringtheir binding capacity. When activated by illumination with light of aparticular wavelength, the fluorochrome-labelled antibody adsorbs thelight energy, inducing a state to excitability in the molecule, followedby emission of the light at a characteristic colour visually detectablewith a light microscope. As in the EIA, the fluorescent labelledantibody is allowed to bind to the first antibody-antigen complex. Afterwashing off the unbound reagent, the remaining tertiary complex is thenexposed to light of the appropriate wavelength and the fluorescenceobserved indicates the presence of the antibody of interest.Immunofluorescence and EIA techniques are both very well established inthe art and are particularly preferred for the present method. However,other reporter molecules, such as radioisotope, chemiluminescent orbioluminescent molecules, may also be employed.

[0030] Further features of the present invention are more fullydescribed in the following Examples. It is to be understood, however,that this detailed description is included solely for the purposes ofexemplifying the present invention, and should not be understood in anyway as a restriction on the broad description of the invention as setout above.

[0031] In the accompanying drawings:

[0032]FIG. 1 shows the location and number of the CB peptides for the α1chain of human type II collagen and the α1 chain of bovine type IIcollagen. CB peptides have been numbered using chick collagen as thestandard. Arrows represent methionine residues in the human type IIsequence and the bovine type II sequence which are the sites of cleavageby cyanogen bromide.

[0033]FIG. 2a shows a carboxymethylcellulose (CM-cellulose) chromatogramand corresponding Coomassie blue protein stain illustrating theseparation of the CB peptides of bovine type ii collagen, using a linearsalt gradient ( . . . ).

[0034]FIG. 2b shows Coomassie blue stained gels of purified fractionsfrom the CM-cellulose column containing various CB peptides, includingCB10 and CB11, of bovine type II collagen.

[0035]FIG. 3 shows the levels of IgG antibodies to human type IIcollagen in patients with rheumatoid arthritis (RA), psoriatic arthritis(PsA), and osteoarthritis (OA), and in normal human sera (NHS). Resultsare expressed as the number of standard deviations (SD) above the meanlevel of antibody in normal sera. A serum is regarded as giving apositive test result if the level of antibody is greater than 3 SD abovethe mean (—). The thick dashed line represents the mean level ofantibody for each group of patients.

[0036]FIG. 4 shows the levels of antibodies to the CB10 peptide ofbovine type II collagen in patients with rheumatoid arthritis (RA),psoriatic arthritis (PsA), and osteoarthritis (OA), and in normal humansera (NHS). Results are expressed as the number of standard deviation(SD) above the mean level of antibody in normal sera. A serum isregarded as giving a positive test result if the level of antibody isgreater than 3 SD above the mean (—). The thick dashed line representsthe mean level of antibody for each group of patients.

EXAMPLE 1

[0037] A. Techniques Used

[0038] Standard Method of Purification of Peptides of Collagen

[0039] Human and bovine type II collagen was prepared from cartilage bypepsin digestion and differential salt precipitation (Rowley, et al.,1988). Bovine type II collagen was digested with cyanogen bromide (CNBr)and the resultant fragments of type II collagen, known as CB peptides,were purified according to the method of Miller and Lunde (1973) withslight modifications; 1.5 g of bovine type II collagen was dissolved ina minimum volume of 70% (v/v) formic acid, containing 1.5 g of CNBr(Sigma, St Louis Mo., USA) and cleavage was achieved by the method ofScott and Veis (1976).

[0040] The CB peptides were initially resolved into 2 major fractions bychromatography on a 2.4×82 cm column of Bio-Gel P-2 (100-200 mesh,exclusion limit 2600 daltons, globular proteins, Biorad, USA)equilibrated with 0.1 M acetic acid. The peptides obtained aftercyanogen bromide digestion were dissolved in 12 ml of 0.1 M acetic acidand 3 ml (˜500 mg) was applied to the column at a time. The largerpeptides, eluted in the excluded volume of the P-2 column, were pooled,lyophilized, and rechromatographed on a 2.5×12 cm cation exchange columnof carboxymethylcellulose (CM-cellulose, Whatman CM-52). The column wasequilibrated with 0.02 M sodium citrate, pH 3.6 containing 0.01 M NaCl,designated as the start buffer, and the peptides were dissolved in thisstart buffer for application to the column. Chromatography was performedat 42° C. over 10 hours using a linear salt gradient obtained by theaddition of 1% 0.16 M NaCl in 0.02 M sodium citrate, pH 3.6, to thestart buffer every 6 minutes, with a flow rate of 1.75 ml/min, using theBiorad Econo system (Biorad, USA). Fractions of ˜17 ml were collected at10 minute intervals, and were run on 15% tricine gels and stained with0.2% (w/v) Coomassie Blue (Biorad, USA). Fractions containing CB10identified by comparison with published type II CB peptide maps(Bornstein et al, 1980; Miller et al, 1982) were pooled, as werefractions containing CB11, desalted on the Bio-Gel P-2 column andlyophilized. CB10 was purified further, and in particular separated fromCB8 and CB9,7, and CB11 was separated from CB12, by applying eachfraction to a 1.0×75 cm column of P-30 (100-200 mesh, exclusion limit 40kD, globular proteins, Biorad, USA) equilibrated in 0.1 M acetic acid.The purity of the peptides after each column run was determined byseparation on 15% tricine gels and staining with 0.2% (w/v) Coomassieblue (FIG. 2a, 2 b).

[0041] Purified CB10 and CB11 were renatured by step-wise cooling from20° C. to 0.2° C., as described by Terato et al, 1985.

[0042] Measurement by ELISA of Antibodies to Native Type II Collagen

[0043] Antibodies to native human type II collagen, and native bovinetype II collagen, were measured by an enzyme linked immunosorbent assay(ELISA). Microtitre plates (Dynatech, Germany) were coated with 100μl/well of 10 μg/ml human or bovine type II collagen overnight at 4° C.Plates were washed 3 times in 10% (w/v) skim-milk powder in PBS, pH 7.4(SM-PBS) containing 0.05% Tween 20 (Sigma, USA) and 3 times in distilledwater (dH₂O), blocked with 1% SM-PBS, 0.05% Tween 20 for 2 hours at roomtemperature, then washed again as above. Sera were tested in duplicateat a dilution of 1:50 in the presence or absence of antigen. Antibodieswere detected using horseradish peroxidase conjugate (HRP) anti-humanIgG (Silenus, Hawthorn, Australia), with 0.5 mg/ml2,2-azino-di-[3-ethyl-benzthiazoline sulfonate (6)] (ABTS) (Boehringer,Germany) in 0.03 M citric acid, 0.04 M Na₂HPO₄, 0.003% H₂O₂, pH 4 assubstrate. Plates were read after 30 minutes at 415 nm on a Bioradplatereader (Biorad, USA). Non-specific binding of immunoglobulins tothe plates was allowed for by subtracting the optical density (OD)obtained in the absence of collagen from that obtained in wells coatedwith collagen.

[0044] Results for test sera were expressed as the number of standarddeviations (SD) above the mean level of antibody for normal blood donorcontrols tested in the same assay. A serum was considered to contain araised level of antibodies to collagen, hereafter specified as positivefor antibodies to collagen, if the level was greater than the mean +3 SDof a panel of 10 blood donor sera tested in the same assay, i.e. if thelevel of antibodies was >3.

[0045] Sera from two patients with high levels of antibodies to nativeand denatured type II collagen, were used throughout as positivecontrols.

[0046] Measurements by ELISA of Antibodies to Purified CB Peptides ofType II Collagen

[0047] Antibodies to the purified CB 10 peptide of bovine type IIcollagen were measured by ELISA as described above for intact type IIcollagen with the following modifications. Plates were coated with 100μl of 10 μg/ml of purified CB10 peptide and sera were tested at adilution of 1:50. The peptide antigen was coated onto the platesovernight at 4° C., blocked with 10% SM-PBS, 0.05% Tween 20 for 90minutes at room temperature with shaking, and sera were incubated for 2hours at room temperature with shaking. Plates were washed 3 times intris buffered saline (TBS) containing 0.05% Tween 20 using an automaticplate washer (Wallac, Finland) in all steps prior to the addition of thesecondary antibody and in distilled water) immediately before and in allsteps subsequent to the addition of the secondary antibody. Boundantibodies were detected using HRP-conjugated anti-human IgG (Silenus,Hawthorn, Australia), and the appropriate substrate. Results areexpressed in terms of the number of standard deviations above the meanfor controls as described above.

[0048] B. Results

[0049] Antibodies to Type II Collagen

[0050] Antibodies to intact human type II collagen measured by ELISAwere present in sera of 25% of 96 patients with rheumatoid arthritis. Bycontrast, antibodies to collagen were present in 15% of 34 patients withosteoarthritis and in 15% of 33 patients with inflammatory arthritisassociated with psoriasis (psoriatic arthritis), representing othernon-autoimmune articular diseases. These figures are comparable withthose reported by others for rheumatoid arthritis and other articulardisease using an ELISA in a similar format (Choi et al., 1988; Fujii etal., 1992). The levels as well as frequencies of antibodies to type IIcollagen were generally higher in rheumatoid arthritis than in otherdiseases (FIG. 3).

[0051] Antibodies to Collagen CB10 Peptide

[0052]FIG. 4 compares the frequency of antibodies tested in a similarELISA in which CB10 peptide prepared from bovine type II collagen wasused as antigen. Antibodies to the CB10 peptide of bovine type IIcollagen were present in the sera of 88% of the same 96 patients withrheumatoid arthritis, but in 6% of patients with osteoarthritis and in12% of patients with psoriatic arthritis. In other words, thereplacement of the intact type II by the CB10 peptide greatly increasedthe sensitivity of the assay for the diagnosis of rheumatoid arthritis,from 25% to 88%, without any decrease in specificity, since thefrequency of positive reactions to the CB10 peptide in the sera frompatients with diseases of the joint other than rheumatoid arthritis wasno greater than the frequency of positive reactions to intact type IIcollagen, i.e. less than 15%.

[0053] This increased sensitivity was shown not to be due to the use ofbovine (versus human) CB10 peptide of collagen in the assay, since thefrequency of reactivity by ELISA with intact collagen is very similarwhether bovine collagen or human collagen is used as antigen in theELISA (see Table I below). In addition, there was substantialspecificity to the reactivity against CB10 since reactivity observed inrheumatoid arthritis with the CB11 peptide, a peptide of collagen ofvery similar size, was only 50%. TABLE 1 Antibody to Antibody to intactintact Antibody to Antibody to human type bovine type bovine bovineGroup No. II collagen II collagen CB10 CB11 Rheumatoid arthritis 96 24(25%) 23 (24%) 84 (88%) 48 (50%) Osteoarthritis 34  5 (15%)  3 (10%)  2(6%)  7 (21%) Psoriatic arthritis 33  5 (15%)  9 (27%)  4 (12%)  2 (6%)Normal human serum 93  0  1 (1%)  3 (3%)  2 (2%)

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1. A kit detecting anti-collagen antibody in a biological sample,wherein said antibody is associated with rheumatoid arthritis,comprising (i) an antigenic preparation comprising the CB10 peptide ofmammalian type II collagen or an antibody-binding fragment of saidpeptide and (ii) means for detecting a complex formed between saidantigenic preparation and antibody from said sample.
 2. A diagnostic kitfor the detection of antibodies to collagen in a biological sample froma patient, comprising, in compartmental form, an antigenic preparationcomprising the CB10 peptide of mammalian type II collagen, or anantibody-binding fragment or variant thereof, and means for detection ofa complex formed between said antigen and antibodies to collagen in saidbiological sample.